Pages: 303-310 DOI: 10.13057/biodiv/d190141
Phylogenetic analysis of colubrid snakes based on 12S rDNA reveals distinct lineages of Dendrelaphis pictus (Gmelin, 1789) populations in
Sumatra and Java
FITRA ARYA DWI NUGRAHA1, FATCHIYAH FATCHIYAH1, ERICNELSON SMITH2, NIA KURNIAWAN1,♥
1Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Brawijaya. Jl. Veteran, Malang 65145, East Java, Indonesia.
Tel.: +62-341-554403, ♥email: [email protected], [email protected]
2Department of Biology, University of Texas at Arlington. Arlington, TX 76019, USA Manuscript received: 17 June 2017. Revision accepted: 25 January 2018.
Abstract. Nugraha FAD, FatchiyahF, SmithEN, Nia Kurniawan N. 2018. Phylogenetic analysis of colubrid snakes based on 12S rDNA reveals distinct lineages of Dendrelaphis pictus (Gmelin, 1789) populations in Sumatra and Java. Biodiversitas 19: 303-310. The phylogenetic relationship among the major colubrid snakes, particularly those of the subfamily Colubrinae, has been the subject of much debate. Also, there was limited data on the molecular relationships of Sundaland colubrid snakes. This study aimed to examine the relationships among colubrid snakes from Sumatra and Java based on fragments of 12S rDNA gene. We sequenced 17 specimens of colubrid snakes representing 5 genera and 2 subfamilies: Colubrinae and Ahaetullinae. We used maximum likelihood, maximum parsimony and Bayesian inference methods for inferring phylogenetic relationships. The result of our phylogenetic analyses is in line with the previous findings for the separation between Colubrinae and Ahaetullinae. Interestingly, we found two distinct clades of Dendrelaphis pictus species with the high genetic divergence between them where D. pictus from Sumatra and West Java separated from Central and East Java clade. Our divergence time estimation showed that the differentiation between these clades of D. pictus occurred in the late Miocene epoch (8.9 Ma) when Sumatra and Java separated after being inundated in the early Miocene epoch.
Keywords: Colubridae, mtDNA, phylogeny, p-distance, Sundaland
INTRODUCTION
Indonesia is known as one of the top biodiverse countries in the world. It comprises about 17,000 islands of various sizes and geological origins (Mittemeier et al.
1999). Based on the Indonesian Biodiversity Action Plan, 16% of the amphibians and reptiles of the world occur in Indonesia (BAPPENAS 1993). The distribution of herpetofauna within those thousands of islands was affected by the geological activities occurred in the past.
These geological activities include volcanic activities, subduction of the Indian ocean crust, and collision of Australia-East Indonesia, which resulted in the rise and widening of Sumatra island at 15 Ma - mid-Miocene.
Another region, East Java-West Sulawesi-Sumba, South West Borneo - rifted from the Australia continental block and were added to Sundaland at approximately 145 Ma - Cretaceous (Hall 2013). The Malay Peninsula, Sumatra, Java and Borneo islands were later connected due to decreasing of the sea level to approximately 118 m below the present sea level during ice ages, most recently at 20,500 - 14,000 years ago (Solihuddin 2014).
Previous studies on animal compositions have revealed complexity in their distribution, in particular within the Sundaland region. Snake populations on the Malay Peninsula and Sumatra are more closely related to each other than to those from Java or Borneo. However, snakes
on the Malay Peninsula, Sumatra, Java, and Borneo were more closely related than they were to those from Sulawesi (Inger and Voris 2001). Based on a simple spatial proximity similarity model, mammals on Mentawai island and Borneo were more closely related to each other than they were to those from Sumatra, the Malay Peninsula and Java, while reptiles and amphibians on those two islands were closely related to those on Sumatra than to those from the other areas (Wilting et al. 2012). Outside of the Sundaland region, several studies have been conducted on colubrid snakes to resolve their taxonomy issues, evolutionary history, biogeographies, and phylogenies (Guo et al. 2012; Lawson et al. 2005; Pyron et al. 2011b;
Vidal et al. 2000; Vidal and Hedges 2002; Zaher et al.
2009). Nevertheless, the phylogeny of Colubridae has not yet been fully resolved, particularly within the paraphyletic Colubrinae (Figueroa et al. 2016; Pyron et al. 2013a).
Unfortunately, there have been few genetic samplings of Colubridae from Java and Sumatra (Shaney et al. 2016).
However, some species of colubrid snakes in Indonesia intrigued some scientists in the past (Auliya 2002; How et al. 1996; van Rooijen and Vogel 2008a, b; 2010; 2012;
Vogel 2008; Vogel and van Rooijen 2007; 2008). Here, we conducted the first study to address the relationship of colubrid snakes within Java and Sumatra islands using 12S rDNA with focusing on Colubrinae and the newly recognized subfamily (Figueroa et al. 2016), Ahaetullinae.
MATERIALS AND METHODS Specimen collection and ethical clearance
This study analyzed eleven specimens of the genus Dendrelaphis from different locations in Sumatra and Java (Figure 1) consisting of D. pictus (Gmelin, 1789), D.
caudolineatus (Gray, 1834), D. haasi van Rooijen &
Vogel, 2008, and D. subocularis (Boulenger, 1888), one specimen of Boiga dendrophila (Boie, 1827) from Central Java, three specimens of the genus Ptyas from central and east Java, one specimen each of Lycodon capucinus (Boie, 1827) and of Coelognathus radiatus (Boie, 1827) from Central and East Java, respectively. Additional colubrid and non-colubrid outgroup sequences were obtained from GenBank (Table 1). To focus the assessment of sequence divergence and relationship on colubrid of the Sundaland region, we restricted our GenBank sampling to only the sequences that came from Sundaland. Therefore, many sequences from the same genera were not included in our analysis because they come from outside of Sundaland. To collect our specimens, we obtained ethical clearance with a letter of Ethical Clearance No: 68-KEP-UB.
DNA isolation, PCR and sequencing
Genomic DNA was isolated from muscle tissue using a QIAGEN DNA isolation kit following the manufacturer’s instruction. A fragment of the 12S gene was amplified by
polymerase chain reaction (PCR) using the primers 12S268 (5’-GTGCCAGCGACCGCGGTTACACG-3’) and 12S916 (5’-GTACGCTTACCATGTTACGACTTGCCCTG-3’) (Jeong et al. 2013). The PCR amplification was performed in 40 µL reaction volumes with 5 min denaturation at 94º C, followed by 35 cycles of 94º C for 30 s, 56,5º C for 1 min, and 72º C for 1 min, and then by post elongation at 72º C for 10 min. The amplified DNA was purified at Biosains Institute, Brawijaya University and sequenced in both directions using the same primers as for PCR by IndoseqGATC.
Sequence and data analysis
DNA sequences were aligned in MEGA 7.0 by the ClustalW method (Kumar et al. 2016). The alignments were edited by eye, with ambiguous sites trimmed (Figueroa et al. 2016). Genetic distances were calculated as uncorrected pairwise distances (Kumar et al. 2016).
Phylogenetic relationships were estimated by maximum likelihood (ML), maximum parsimony (MP) and Bayesian inference (BI). ML analyses were performed with 1000 bootstrap replicates in MEGA 7.0 (Kumar et al. 2016) with Kimura2 parameter model. PAUP 4.0b10 was used to performed MP analyses with 1000 bootstrap replicates (Swofford 2002) and tree bisection recognition (TBR) branch-swapping algorithm on heuristic search option.
Figure 1. Locations of specimens collected from Sumatra and Java, Indonesia and included in this study
Table 1. List of GenBank accession numbers of sequences used in the phylogenetic analysis.
Specimens Locality Country Genbank
accession no. Source Boiga dendrophila Cilacap, Central Java Indonesia KY700852 This study Coelognathus radiatus Malang, East Java Indonesia KY700853 This study
Dendrelaphis caudolineatus Aceh Indonesia KY700854 This study
Dendrelaphis caudolineatus Bengkulu Indonesia KY700855 This study
Dendrelaphis sp. Sukabumi, West Java Indonesia KY700856 This study
Dendrelaphis subocularis Bogor, West Java Indonesia KY700857 This study
Dendrelaphis haasi Bengkulu Indonesia KY700858 This study
Dendrelaphis pictus Bogor, West Java Indonesia KY700859 This study
Dendrelaphis pictus South Lampung, Lampung Indonesia KY700860 This study
Dendrelaphis pictus Aceh Indonesia KY700861 This study
Dendrelaphis pictus Medan, North Sumatra Indonesia KY700862 This study Dendrelaphis pictus South Malang, East Java Indonesia KY700863 This study Dendrelaphis pictus Wonosobo, Central Java Indonesia KY700864 This study
Lycodon capucinus Sadang, Central Java Indonesia KY700865 This study
Ptyas korros Cilacap, Central Java Indonesia KY700866 This study
Ptyas korros Cilacap, Central Java Indonesia KY700866 This study
Ptyas mucosa South Malang, East Java Indonesia KY700867 This study
Boiga cynodon Thailand Thailand Z46468 Heise et al. 1995
Boiga forsteni Sri Lanka Sri Lanka KC347314 Pyron et al. 2013b
Lycodon laoensis Suphanburi Thailand Z46455 Heise et al. 1995
Coelognathus radiatus Thailand Thailand AY122676 Utiger at el. 2005
Coelognathus flavolineatus Java Indonesia AY122666 Utiger at el. 2005
Coelognathus erythrurus Sibutu island Philippines AY122776 Utiger at el. 2005
Ptyas mucosa Kathmundu, Nepal India AY122828 Utiger at el. 2005
Dendrelaphis caudolineatus Thailand Thailand AF544782 Vidal and Hedges 2002
Enhydris enhydris - - EF395879 Alfaro et al. 2008
Hypsiscopus plumbea - - EF395884 Alfaro et al. 2008
Naja naja - - AF236683 Unpublished
Transition and transversion were equally weighted and the gaps were treated as missing data. BI was calculated with MrBayes 3.0b4 (Huelsenbeck and Ronquist 2001).
The analysis performed under 4 simultaneous metropolis couple Monte Carlo Markov Chains (MCMC) for 1,000,000 generations and sampled a tree in every 1000 generations. The consensus topology was calculated by discarding the tree with the burn-in of 25%.
The divergence time was estimated with BEAST (Drummond and Rambaut 2007) under a Hasegawa- Kishino-Yano (Hasegawa et al. 1985) model of DNA evolution with uncorrelated log-normal relaxed clock rate model (Drummond et al. 2006). The external calibration used the time divergence between Homalopsidae, Colubridae and Elapidae at 49.2 Million years ago (Ma) (CI: 39-61); between Colubridae and Elapidae at 46.3 Ma (CI: 36-58) (Vidal et al. 2009); and between Enhydris enhydris and Enhydris plumbea at 18.9 Ma (CI: 12.9-24.9) (Alfaro et al. 2008). The internal calibration used the divergence time of Colubridae at 36.6 Ma (CI: 28-46) (Vidal et al. 2009); and between Ptyas mucosa and Ptyas korros at 21 Ma (Nagy et al. 2004). The analysis used MCMC chain every 1000 generations, for a total of 10,000,000 samples and assessed to the stationary distribution through inspection of the likelihood and parameter sample plots in Tracer v1.6 (Rambaut et al.
2014). All of the phylogenetic trees from the analyses were visualized with Figtree v1.4.2 (Rambaut 2014).
RESULTS AND DISCUSSION Genetic Distances
The uncorrected pairwise distance or p-distance is described as the difference of nucleotide which being compared (Nei and Kumar 2000). Regardless of the locality, p-distance among Boiga ranged from 5.6% to 6.5% (mean±sd; 5.99 ± 0.47). Genetic distances were in the genus of Coelognathus (mean±sd;6.40 ± 3.50), which ranged from 4.0% to 9.0%. Sequence divergence within Ptyas ranged from 0.62% to 7.4% (mean±sd;5.06 ± 3.68).
Within Dendrelaphis, p-distance values ranged from 5.9%
to 11.8% (mean±sd; 6.36 ± 3.79) (Table 2). The intra- species threshold for 12S/16S was previously set at 3%
(Jeong et al. 2013). Based on our result, we propose that 12S threshold for intra-species is 3.7%, inter-species is 11.8% and inter-generic is 16%. Our results showed the higher value of sequence divergence than the previous studies which suggests that the colubrid snakes in Java and Sumatra are genetically more diverse than those of other places.
Interestingly, D. pictus of West Java showed a greater distance when it compared to Central or East Java (3.1- 3.72%) than to Sumatra (0.31-0.93%). The value of p- distance showed that D. pictus from Sumatra and West Java were genetically distinct from those in Central and East Java (Table 3).
Table 2. Uncorrected p-distances (%) for 12S rDNA among different genera of colubrid snakes of Java and Sumatra
Genus Mean ± SD Range
Boiga 5.99 ± 0.47 5.6-6.5
Coelognathus 6.40 ± 3.50 4.0-9.0
Ptyas 5.06 ± 3.68 0.62-7.4
Dendrelaphis 6.36 ± 3.79 5.9-11.8
Table 3. Uncorrected p-distances (%) for 12S rDNA of Dendrelaphis pictus from different localities of Java and Sumatra
No. Locality 1 2 3 4 5
1 Aceh, Sumatra
2 Medan, North Sumatra 0.3 3 Lampung, Sumatra 0.6 0.3 4 Bogor, West Java 0.9 0.6 0.3 5 Wonosobo, Central Java 3.7 3.4 3.7 3.1 6 Malang, East Java 3.4 3.1 3.4 3.4 0.3
Relationships among Colubridae in Sumatra and Java islands
The ML, BI and MP trees showed identical topology in the case of separation between the subfamilies Colubrinae and Ahaetullinae. In the monophyly of Colubrinae, the group of Ptyas found to be paraphyletic, while within Ahaetullinae, D. pictus was separated from D.
caudolineatus (Figure 2). Monophyly of Colubrinae was strongly supported in all trees (Figure 2), while in
Ahaetullinae it was strongly supported in the BI and MP trees (BPP=1, MP=70) yet weakly in the ML tree.
In the subfamily of Colubrinae, two P. korros from Cilacap formed a clade and was strongly supported in all trees. In the ML and BI trees, this clade was separated from other remaining species consisting of P. mucosa, Coelognthus, Lycodon and Boiga, while P. mucosus from East Java and P. mucosa from India were in a clade which formed a sister to the clade consisting of Coelognathus, Lycodon, and Boiga. The group of Coelognathus formed a clade (BPP=0.93) where C. radiatus from East Java and C.
radiatus from Thailand (ML=100, BPP=1, MP=100) separated from C. erythrurus and C. flavolineatus (ML=84, BPP=1, ML=60). The Colubrinae genus Coelognathus clade was separated from the clade consisting of Lycodon and Boiga, where the clade of Lycodon formed a sister to the clade of Boiga. The species of L. laoensis formed a clade with L. capucinus (ML=89, BPP=1, MP=87) and B.
dendrophila nested in a clade with B. forsteni in the ML and BI trees, but with B. cynodon in the MP tree.
Within Ahaetullinae, D. caudolineatus from Aceh, Bengkulu, and Thailand and Dendrelaphis sp. from Sukabumi formed a clade with significant bootstrap values in all trees. The clade consisting of D. subocularis, D.
haasi, and D. pictus was divided into two subclades. The first subclade consists of D. subocularis from Bogor and D.
haasi from Bengkulu, which was a sister clade to D. pictus from Bogor, Lampung, Medan, and Aceh. The second subclade consists of D. pictus from Wonosobo and Malang (ML=93, BPP=1, MP=96).
Figure 2. Phylogeny of Colubridae in Sumatra and Java islands based on partial sequence of 12S rDNA. Left: ML and Bayesian inference tree. Nodal supports represent ML bootstrap value/Bayesian posterior probability (BPP) (-) means no value. Right: MP tree with the bootstrap values on the branches
The present study is consistent with previous studies by Pyron et al. (2011; 2013a, b) and Figueroa et al. (2016) which separated Dendrelaphis from the Colubrinae group.
Based on their results, Figueroa et al. (2016) proposed a new subfamily Ahaetullinae, which consists of Ahaetulla, Chrysopelea, Dendrelaphis, and Dryiophiops and covers 56 species. B. dendrophila (Cilacap, Central Java) in this study formed a clade with B. forsteni (Sri Lanka) and B.
cynodon (Thailand), which is consistent with the subfamily proposed by Pyron et al. (2013a, b). However, Figueroa et al. (2016) showed that B. dendrophila formed a clade with B. cyanea, which was a sister clade to B. forsteni and B.
cynodon. Additionally, two specimens of P. korros (Cilacap, Central Java) and P. mucosus (Malang, East Java) did not form a clade as showed by Utiger et al. (2005) and Pyron et al. (2011; 2013a), yet comprised a sister clade to Coelognathus (Utiger et al. 2005).
Dendrelaphis formed a clade with Oreocalamus (Kraus and Brown 1998). An individual of D. caudolineatus, Ahaetulla fronticincta, Chrysopelea paradisi formed a clade and became a sister clade to all remaining colubrid snakes (Pyron et al. 2011). Four single-specimens of Dendrelaphis formed a clade; D. caudolineatus formed a clade with D. schokari, D. tristis and D. bifrenalis (Pyron et al. 2013a), while in Pyron et al. (2013b) D. bifrenalis formed a clade with D. pictus and D. calligaster. The study with more comprehensive specimens and genetic sampling showed that D. caudolineatus separated from D. haasi, D.
cyanochloris, and D. pictus, which were nested in a paraphyletic clade (Figueroa et al. 2016). In contrast to our expectation, D. pictus was found to be paraphyletic, with separation of the clade between Sumatra-west Java and central-east Java. Given that the high p-distance value and separated clade of the D. pictus group, we consider the geological history which could influence on the genetic differentiation in this area below; while the taxonomic status of this snake remains an area for further investigation.
The opportunities for gene flow among snakes from Sumatra, Java and Borneo would have been possible until the end of the Oligocene (±25 Ma) and again at the end of Miocene (±10 Ma). The exposed continents of Sundaland during much of Pleistocene were partially covered by savanna or grasslands. It provided the corridor for the migration of the snakes from the north of Sundaland to the south in Java region (Inger and Voris 2001). The prehistoric environmental conditions also had an enormous influence on the divergence of these snakes (Allam and Abo-Eleneen 2012). However, based on the map of Heaney (1991), the Pleistocene savanna or grasslands only covered part of the area between Sumatra-Borneo and Borneo- Eastern Java. It would have led the snakes to come only to East part of Java and then diversified genetically from those in Western Java and Sumatra. Unfortunately, our work did not include D. pictus from Borneo, which might be critical to understanding the pattern of distribution.
Historically, the east part of Java was a block rift from continental Australia, as along with SW Borneo and West Sulawesi. The blocks were added to Sundaland at approximately 90 Ma from West Java through Meratus mountains in the north (Hall 2014). According to this, snakes would have existed in these blocks since the snakes arose at approximately 125 Ma and they have become a distinct population from those in West Java. However, this scenario is not supported by the oldest known fossil of colubrid snake found in Wai Lek, Thailand, which was considered to date from the late Eocene (Rage et al. 1992).
Moreover, we still consider this scenario as the likely speciation process between Sumatra-west Java and Central- East Java population due to limited fossil information, particularly for D. pictus.
The divergence time of Colubrid snakes
Our analysis on divergence time estimated that Colubrid snakes started to diverge in the late Eocene approximately 37.6 Ma. It was the period when most of western of Sundaland formed a connected landmass with many large freshwater lakes; while southern Sundaland comprised volcanic arcs that formed new islands. This age was considered as the collision time between India and Asia (Hall 2013), which might have facilitated many migrations of terrestrial fauna. From late Eocene, colubrid snakes began to spread from Asia to Europe by Oligocene, to North America through Beringia and to the west of America (Holman 1984). Moreover, colubrid rat-snakes began to spread from the eastern Palearctic to the western Palearctic and Nearctic at about 20 Ma (Chen et al. 2017).
The result was also in line with the oldest Colubridae fossil in Wai Lek pit, which was dated from the late Eocene (Rage et al. 1992). The subfamily Colubrinae began to diversify in early Miocene at about 25 Ma, when rainforest extended to the north over much of Sumatra and when shallow seas inundated Java while Ahaetullinae diversified later in the Miocene, at about 24 Ma. D. caudolineatus began to diverge in the late Miocene at about 11.3 Ma, while genetic differentiation among D. pictus populations from Sumatra-West Java and Central-East Java occurred later at about 9.0 Ma when many small volcanic islands emerged in Sumatra and Java (Hall 2013).
Although the diversification of colubrids was estimated to have started in the Eocene epoch, our relaxed normal clock showed that most of the inter-specific divergence occurred in the Miocene epoch, when the majority of Java and Sumatra was inundated by a shallow sea. Glaciation in the Pleistocene epoch caused lowering of the sea level, which formed a continuous landmass of Sundaland.
Savannah and open woodlands that grew up in this region may have facilitated a huge spread of colubrid snakes. To address the distinct lineage of D. pictus between Sumatra- West Java and Central-East Java, we discussed three possibilities that caused the distinction.
Figure 3. The divergence chronogram of colubrid snakes of Sumatra and Java based on 12S rDNA. The tree was constructed based on a relaxed normal clock and Bayesian inference with 95% credible interval
Table 4. The time of divergence of Colubridae with 95% credible interval
Clade Divergence time
(Ma) 95% CI
(Ma)
1 49.3 45.488-54.977
2 47.2 40.466-51.489
3 37.6 33.419-43.257
4 25.1 21-30.204
5 24.8 18.279-32.931
6 23.1 20.093-26.669
7 20.9 19.326-23.076
8 20.7 15.669-26.46
9 19.6 13.558-24.105
10 17.9 14.581-21.5
11 13.1 8.163-18.722
12 11.3 10.455-17.787
13 10.5 6.831-15.094
14 9.9 5.094-15.281
15 9.2 4.53-11.854
16 9.0 5.634-13.334
17 8.4 4.742-11.314
18 6.0 2.817-8.615
19 1.8 0.564-3.545
20 1.4 0.094-3.075
21 1.2 0.07-2.019
22 1.2 0.141-2.653
23 0.9 0.094-2.09
24 0.8 0.07-1.784
25 0.6 0.07-1.432
26 0.6 0.094-0.986
27 0.4 0.094-1.286
First, if Rage (1992) is correct about the Asiatic origin of Colubridae, the migration through a land bridge for D.
pictus must come from South of Java which is Borneo or Sumatra and began to diversify. The possibility to migrate occurred only in the Pleistocene epoch because there was no a land bridge which connected these islands since Eocene through Miocene. However, it was not corroborated by the divergence time data. The Pleistocene began at 1.8 Ma, while the separation of D. pictus populations in Sumatra-west Java from those in Central- East Java evidently occurred at 9 Ma. Second, West Sulawesi and East Java were continental blocks that rifted from Australia and were added to Asia in the Cretaceous period (Hall 2014). If D. pictus from Central-East Java came from these continental blocks, Colubridae should have appeared in the Jurassic period or at least in the Cretaceous, but the oldest fossil Colubridae was considered only from the late Eocene which is very far from Jurassic.
Hence, the two possibilities described above are contradicted by other data and cannot explain the high genetic differentiation between D. pictus populations of Sumatra-West Java and Central-East Java.
The last possibility uses the migration theory proposed by Inger and Voris (2001), material rafting. When D. pictus reached the west part of Java, the volcano activities caused migration of the snakes down from their habitat, they randomly attached to material that floats on the water, and the sweepstakes migration followed the wind or cyclonic
storm to the central part and east part of Java. The scaly epidermis may have facilitated and protected them from crossing the saline water. Once D. pictus reached central or east part of Java, the gene flow was obstructed between the regions of Sumatra-West Java with Central-East Java.
However, the possibility of inhibition of gene flow only lasted until the Ice Age in the Pleistocene, because in this epoch the distribution of snakes becomes larger than the previous wave of distribution in the final epoch of the Eocene, Oligocene, Miocene or Pliocene. This happened due to the lowering sea level and the growth and development of savanna in the mainland of Sundaland. In addition, the river system linking the island of Borneo to Central and East Java, as well as West Java with the central and eastern part also provided a very suitable corridor for the migration of D. pictus to and from those areas. As a result, the gene flow that had been interrupted in the previous epoch (Eocene-Pliocene), which was not strong enough to make D. pictus in the West Java significantly different from Central and East Java, became reopened in the Pleistocene epoch. Hence, the gene flow could still occur again among populations of D. pictus of western Java with those in central and eastern Java.
This study showed that geological history and ecological variation of Sumatra and Java facilitated the distribution and the diversification of colubrid snakes in those islands. Moreover, our study has found evidence that East and Central Java has a unique diversity compared to West Java and Sumatra as it found in L. hasseltii (Hamidy and Matsui 2017). However, the present study should be confirmed with further studies using more genes and more comprehensive methods, since we only used one mitochondrial gene in this study. More specimens from adjacent islands such Bali, Madura, Borneo, and Sulawesi are also crucial to reveal the origin of East Java diversity, especially that of D. pictus.
ACKNOWLEDGEMENTS
Field sampling was financially funded by the NSF to Eric N. Smith (NSF Award Number: DEB-1146324) and PEER SCIENCE USAID grant to Nia Kurniawan (Sub- grant number: 2000005071). Molecular work was supported by Indonesian LPDP to Fitra Arya Dwi Nugraha (Number: PRJ-4450 /LPDP.3/2016). We acknowledge A.M. Kadafi, B. Priambodo, and A.S. Firdaus for collecting all specimens used in this study, M. Fahmi for CIPRES online and PAUP analysis, Adityas and A.S.
Kurnianto for sharing discussions.
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